Inhibition of intracellular signal transduction by 14-3-3-binding peptides

ABSTRACT

Compositions and methods for inhibition of the intracellular activation of a signal transducing protein comprising phosphoserine containing peptides derived from the Raf-1 protein are disclosed. The peptides bind to 14-3-3 protein and thereby prevent its association with the signal transducing peptide to block signal transduction. Also provided is an assay method for identifying a pharmacologic agent that can bind 14-3-3 protein and block signal transduction.

This is a divisional of application Ser. No. 08/616,669; filed on Mar.15, 1996, now U.S. Pat. No. 5,948,765.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

This invention relates to intracellular proteins involved in signaltransduction and, more particularly, to peptides and methods forblocking signal transduction by binding to 14-3-3 proteins.

(2) Description of the Related Art

The family of proteins known as 14-3-3 proteins are expressed in a widevariety of organisms and tissues and at least seven different isoformsexist in mammalian cells (Aitken et al., Trends Biochem Sci 17:498-501,1992; Aitken, Trends Biochem Sci 20:95-97, 1995 which are incorporatedby reference). The 14-3-3 proteins appear to mediate a number ofbiological activities. This protein has been found to activate neuronaltyrosine and tryptophan hydroxylase (Ichimura et al., Proc Natl Acad SciUSA 85: 7084-8, 1988 which is incorporated by reference); to regulatethe activity of protein kinase C (Isobe et al. FEBS Letters 308:121-124,1991; Toker et al. Eur J Biochem 191: 421-9, 1990; Tanji et al., J.Neurochem 63: 1908-16, 1994 which are incorporated by reference); aswell as to bind to and presumably regulate a number of signalingproteins including Raf-1, polyoma middle T antigen, bcr, and PI-3 kinase(Fantl et al., Nature 371: 612-4, 1994 which is incorporated byreference).

One of these proteins, Raf, constitutes a family of serine/threoninekinases involved in the transduction of signals for growth anddevelopment from the cell surface to the nucleus. Members of the Raffamily of kinases include Raf-1 which is ubiquitously expressed andA-Raf and B-Raf which have restricted patterns of expression. Rafkinases are believed to be key mediators of mitogenesis anddifferentiation, acting through a cascade of protein kinases that isalso thought to be the pathway utilized by most oncogenes in celltransformation. (See Daum et al., Trends Biochem Sci 19:474-480, 1994which is incorporated by reference.) The activation of Raf-1 in thoughtto involve phosphorylation of the molecule and several phosphorylationsites have been identified. (Morrison et al., J Biol Chem 268:17309-16,1993 which is incorporated by reference). The phosphorylated Raf proteinmay then bind to the 14-3-3 protein which has been suggested to beessential to activation of Raf-1 in its mediation of these eventsinasmuch as microinjection of 14-3-3 results in Raf-1 activation and isrequired for function when Raf-1 is expressed in yeast (Fantl et al.,supra; Li et al., EMBO J 14: 685-96, 1995; Irie et al., Science265:1716-1719 which are incorporated by reference). It has beensuggested that binding of 14-3-3 to Raf-1 is not necessary foractivation, inasmuch as another group of investigators have reportedthat mutant forms of Raf-1 that are unable to bind to the 14-3-3 proteinnevertheless show in vitro kinase activity as well as the ability toinduce meiotic maturation in oocytes thus suggesting that binding to14-3-3 is not essential (Michaud et al., Mol Cell Biol 15:3390-3397,1995 which is incorporated by reference). This work is based upon theidea that 14-3-3 binds to a phosphorylation site (ser-259) that isinduced by growth factor treatment. But 14-3-3 is associated with Raf-1constitutively so this phosphorylation site (ser-259) cannot be the onlybinding site for 14-3-3. Thus, it was not appreciated that 14-3-3 bindsto a Raf-1 phosphorylation site that is essential for function as isdisclosed herein.

Nevertheless, because of the role of Raf-1 and possibly also the 14-3-3proteins in a disease process involving growth and differentiation, inparticular such conditions as cancer, atherosclerosis and autoimmunedisease, it would be desirable to provide a method for interrupting thissignal transduction pathway and thereby provide a new approach totreating these diseases.

BRIEF DESCRIPTION OF THE INVENTION

Accordingly, the inventors herein have succeeded in devising novelcompositions and methods that act to inhibit the activation of a signaltransducing protein by 14-3-3 protein by binding to the 14-3-3 proteinand thereby prevent its binding to the signal transducing protein andits subsequent activation. Because the 14-3-3 proteins are utilized in aubiquitous manner in the activation of signal transducing proteins, thepresent methods and compositions are applicable to a wide variety ofsuch signal transducing proteins.

In one embodiment, the present invention provides a compositioncomprising an isolated phosphoserine-containing peptide comprising theamino acid sequence Arg-Ser-Xaa₁-Xaa₂-Xaa₃-Pro where Xaa₁ is any aminoacid, Xaa₂ is a phosphorylated serine and Xaa₃ is any amino acid (SEQ IDNO: 1). This sequence is based upon the amino acid residues surroundingboth the Serine-259 (residues 256-261) and Serine-621 (residues of618-623) of Raf-1. More particularly, peptides within the scope of thisinvention can contain the Serine-259 specific amino acid sequenceArg-Xaa₁-Arg-Ser-Xaa₂-Xaa₃-Xaa₄-Pro where Xaa₁ and Xaa₂ are any aminoacid, Xaa₃ is a phosphorylated serine and Xaa₄ is any amino acid (SEQ IDNO: 2) or the Serine 621 specific amino acid sequenceLys-Xaa₁-Xaa₂-Arg-Ser-Xaa₃-Xaa₄-Xaa₅-Pro where Xaa₁, Xaa₂ and Xaa₃ areany amino acid, Xaa₄ is a phosphorylated serine and Xaa₅ is any aminoacid (SEQ ID NO: 3). Peptides containing these sequences specificallybind to the 14-3-3 protein preventing the activation of a protein of theRaf-1 family of kinases that are signal transducing proteins. Othersuitable peptides can be designed containing all or part of the 6 aminoacids of SEQ ID NO: 1 so long as the Xaa₂ is a phosphorylated serine andthe peptides are capable of binding to a 14-3-3 protein that activates amember of the Raf-1 family of signal transducing proteins. Similarly,such other suitable peptides which are capable of binding to a 14-3-3protein that activates a Raf-1 family member can contain all or part ofSEQ ID NO:2 or SEQ ID NO:3 so long as they contain a phosphorylatedserine. Peptides containing 6 amino acids corresponding to SEQ ID NO: 1in which the serine in position Xaa₂ is not phosphorylated show littleor no capacity to bind to isoforms of the 14-3-3 protein.

Both peptide and non-peptide derivatives of the peptides can also beprepared that exhibit the functionality of being capable of binding tothe 14-3-3 protein and blocking the binding of 14-3-3 to the signaltransducing protein to inhibit its signal transducing properties in acell.

In another embodiment, a method is provided for inhibiting theintracellular activation of a signal transducing protein byadministering to a cell an effective amount of a peptide that containsthe amino acids sequences as set forth in SEQ ID NO: 1 or SEQ ID NO: 2or SEQ ID NO: 3 or a derivative thereof wherein the peptide orderivative thereof binds to a 14-3-3 protein. The administering of theexogenous peptide or derivative thereof to a cell and the binding of thepeptide to the 14-3-3 protein blocks the binding of 14-3-3 to the signalprotein resulting in inhibition of the signal transducing activity ofthe protein.

In another embodiment, a method is provided for identifying a substancethat binds to a 14-3-3 protein and thereby inhibits the activity of asignal transducing protein. The method comprises forming a mixturecomprising a construct containing a 14-3-3 protein or derivative thereofimmobilized to a substrate, a labeled peptide containing a sequenceobtained from a Raf-1 sequence surrounding serine-259 or serine-621, andsaid pharmacologic agent. The mixture is then incubated under conditionsunder which the labeled peptide can bind to the construct but for thepresence of the pharmacologic agent. The binding of the labeled peptideis then determined wherein a decrease in binding of the labeled peptideindicates a binding of the pharmacologic agent to the construct. Alsoincluded within the scope of the present invention is the pharmacologicagent identified by this method. The method can also be used to isolatethe pharmacologic agent such as from a library of chemical substances.

Among the several advantages found to be achieved by the presentinvention, therefore, may be noted the provision of a composition whichblocks the effects a growth factor or oncogene by binding 14-3-3 andinactivating or preventing the activation of the signal transducingprotein, Raf-1; the provision of a method for blocking the intracellularactivation of a signal transducing protein by administering to a cell aneffective amount of a composition that binds to 14-3-3 protein; theprovision of a method for identifying and/or isolating a pharmacologicagent that binds to 14-3-3 and inhibits the activation of Raf-1; and theprovision of pharmacologic agents identified and/or isolated by saidmethod.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the binding, measured by scintigraphy, of the Raf-259peptide (SEQ ID NO: 4), which has been radiolabeled and phosphorylated,to immobilized GST/14-3-3 zeta in the absence or presence of differentconcentrations of cold phosphorylated non-radiolabeled wild-type Raf-259peptide (cold phospho-259, SEQ ID NO: 5), non-phosphorylated andnon-radiolabeled wild-type Raf-259 peptide (unphospho-259, SEQ ID NO:4), phosphorylated casein Kinase II substrate peptide, RRREEEpTEEE(phospho-ETEE, SEQ ID NO: 6), non-radiolabeled free phosphoserine(phosphoserine), or the tyrosine-phosphorylated peptide, pYEEISPAK,(phospho-YEEI, SEQ ID NO: 7).

FIG. 2 illustrates the competitive displacement of the phosphorylatedand radiolabeled wild-type Raf-1 peptide (SEQ ID NO: 4) from immobilizedGST/14-3-3 zeta by non-radiolabeled, phosphorylated Raf-259 peptide (SEQID NO: 5) measured by scintigraphy.

FIG. 3 illustrates the surface plasmon resonance assessment of peptidebinding to 14-3-3 protein showing the binding of GST-14-3-3 zeta (1 μM)to phosphorylated biotinylated, Raf-1 peptide bound to a strepavidincoated sensor and lack of binding of the correspondingnon-phosphorylated peptide.

FIG. 4 illustrates the inhibition of dephosphorylation of Raf-1 by14-3-3 showing (A) the phosphorylated GST-Raf fusion protein band on 10%acrylamide SDS gel analyzed by autoradiography (lane 1); absence of thesame band on incubation with the serine/threonine phosphatase, PP1 (lane2); presence of the band indicating inhibition of dephosphorylation inthe presence of 14-3-3 protein (lane 3); and absence of the band withthe control fusion protein GST-p56-lck and (B) the phosphorimagerquantitation of results depicted in panel A.

FIG. 5 illustrates the inhibition of 14-3-3/Raf-1 complex formation invitro detected by immunoblotting using polyclonal anti-Raf-1 showingcomplex formation with GST-14-3-3 zeta immobilized onglutathione-agarose and NIH 3T3 cell lysate (lane 1), no complexformation with GST alone and lysate (lane 2), complex formation withGST-14-3-3 zeta, lysate, and non-phosphorylated Raf-259 peptide (SEQ IDNO: 4) (lane 3) and no complex formation with GST-14-3-3 zeta, lysateand phosphorylated Raf-259 peptide (SEQ ID NO: 5) (lane 4).

FIG. 6 illustrates disruption of pre-existing Raf-1/14-3-3 complexes invitro using phosphorylated Raf-259 peptide (SEQ ID NO: 5: lane 2, 10 μM;lane 3, 100 μM) compared to the unphosphorylated Raf-259 peptide (SEQ IDNO: 4: lane 1, 100 μM).

FIG. 7 illustrates the inhibition of germinal vesicle breakdown (GVBD)in Xenopus oocytes stimulated with insulin after microinjection ofphosphorylated Raf-259 peptide (SEQ ID NO: 5) (pS-259) compared to noinhibition after microinjection of non-phosphorylated Raf-259 peptide(SEQ ID NO: 4) (unphos-259) or microinjection of water (- pS-259 and -unphos-259).

FIG. 8 illustrates the inhibition Raf-1 kinase activity byphosphorylated Raf-259 peptide showing no Raf-1 kinase activity inprotein lysates made from oocytes not stimulated with insulin andmicroinjected with water (lane 1), no Raf-1 kinase activity in lysatefrom insulin-stimulated, water-injected oocytes (lane 2), no Raf-1kinase activity in lysate from insulin-stimulated oocytes injected withphosphorylated Raf-259 peptide (lane 3), and Raf-1 kinase activity inlysate from insulin-stimulated oocytes injected with non-phosphorylatedRaf-259 peptide (lane 4).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, it has been discovered thatsignal transducing proteins, in particular the Raf-1 family of signaltransducing proteins can be inhibited by the administration of acomposition containing a peptide with a phosphorylated serine residue.The composition is capable of binding to the 14-3-3 protein in the cell.Administration of the peptide results in the blocking of the cellulareffect of activation of the Raf-1 signal transducing protein by asubstance such as a growth factor or an oncogene.

The process of identifying and preparing peptides containing aphosphorylated serine residue in which the peptide is capable of bindingto 14-3-3 protein and inactivating a specific signal transducing proteinis exemplified below for the isolation of the peptides that bind 14-3-3and thereby inactivate the Raf-1 family of kinases. By inactivation itis meant that the protein or complex of proteins capable of signaltransduction is either not produced or once produced, it is altered insuch a manner that it is no longer capable of signal transduction.

Although it had been reported that the serine residues at positions 259and possibly also position 621 of Raf-1 may be required for Raf-1 tobind to 14-3-3 protein and that dephosphorylated Raf-1 is unable to bindto 14-3-3 (Michaud et al. supra), nevertheless, the essentialrequirement for the binding of 14-3-3 protein to phosphoserines atposition 259 and more particularly at position 621 of Raf-1 in theactivation of Raf-1 was not appreciated. The present invention is basedupon the heretofore unrecognized direct and specific interaction of14-3-3 protein with phosphoserine-containing peptides derived from aRaf-1 sequence surrounding the serine-259 and, more particularly, theserine-621.

One composition of the present invention comprises a peptide thatincludes a sequence of 6 amino acids that comprisesArg-Ser-Xaa₁-Xaa₂-Xaa₃-Pro where Xaa₁ is any amino acid, Xaa₂ isphosphorylated serine and Xaa₃ is any amino acid (SEQ ID NO: 1). It hasbeen found that a peptide molecule containing this sequence is capableof directly and specifically binding to 14-3-3 protein blockingactivation of Raf-1 signalling. This 6 amino acid sequence correspondsto the sequence of amino acids 256-261 (Arg-Ser-Thr-Ser-Thr-Pro, SEQ IDNO: 8) and amino acids 618-623 (Arg-Ser-Ala-Ser-Glu-Pro, SEQ ID NO: 9)of Raf-1 except that the serine in position 259 or 621 isphosphorylated. By phosphorylating the serine residue in the amino acidsequence of SEQ ID NO: 8 or SEQ ID NO: 9, the phosphorylated peptidebecomes capable of specific binding with 14-3-3 protein in the cell.

The novel peptides within the scope of the present invention can alsocontain the Serine-259, Raf-1 derived amino acid sequenceArg-Xaa₁-Arg-Ser-Xaa₂-Xaa₃-Pro where Xaa₁ is any amino acid, Xaa₂ is aphosphorylated serine and Xaa₃ is any amino acid (SEQ ID NO: 2) or theSerine 621, Raf-1 derived amino acid sequenceLys-Xaa₁-Xaa₂-Arg-Ser-Xaa₃-Xaa₄-Pro where Xaa₁ is any amino acid, Xaa₂any amino acid, Xaa₃ is a phosphorylated serine and Xaa₄ is any aminoacid (SEQ ID NO: 3).

The novel peptides of the present invention can also comprise otherpeptides that are longer than SEQ ID NOS: 1-3 in which additional aminoacids have been added to either of the terminal amino acids so long asthe resulting peptide remains capable of binding to the 14-3-3 protein.Other such exemplary peptides suitable for binding to a 14-3-3 proteinand blocking Raf-1 signal transducing activity include a 15 amino acidpeptide based upon the sequences surrounding serine-259 of Raf-1, i.e.,Leu-Ser-Gln-Arg-Gln-Ser-Thr-Xaa-Thr-Pro-Asn-Val-His-Ala where Xaa is aphosphorylated serine (SEQ ID NO: 5) and the sequences surroundingserine-621 of Raf-1,Leu-Pro-Lys-Ile-Asn-Arg-Ser-Ala-Xaa-Glu-Pro-Ser-Leu-His-Arg where Xaa isa phosphorylated serine (SEQ ID NO: 10). In addition, certain aminoacids with SEQ ID NO: 1 can be replaced with other amino acids so longas the phosphoserine is retained. Preferably, peptides prepared inaccordance with the present invention are between six and about thirtyamino acids in length. The peptides can be derived from the Raf-1molecule or they can be an unrelated sequence provided that the sixamino acid sequence of SEQ ID NO: 1 is included in the peptide or issimilar to the sequence of SEQ ID NO: 1 maintaining that the serine inthe amino acid sequence is phosphorylated.

In addition, modified serine residues such as phosphono-serines orserines with sulfur derivatives can also be used in place ofphosphoserine as well as glutamic acid, which like the phosphoserineresidue is negatively charged.

Included within the scope of peptides of the present invention are alsosequences based upon paralogs and orthologs of Raf-1 and hybrid andmodified forms of the peptides of the present invention in which certainamino acids have been deleted or replaced or modifications such as whereone or more amino acids have been changed to a modified or unusual aminoacid so long as the peptide retains the ability to bind to 14-3-3protein and block the activation of Raf-1 by the 14-3-3 protein.

Based upon the results reported herein, the inventors believe that theSerine-621 of Raf-1 may be a more preferred binding site than theSerine-259 of Raf-1 for 14-3-3 protein when these serines arephosphorylated. Thus, the preferred peptides of the present inventionare based upon the amino acids surrounding the Serine-621 of Raf-1.

The serine residue in the peptide sequences of the present invention canbe phosphorylated by known methods. The peptide also can be produced bystandard synthetic procedures such as by “classical” Merrifield methodof solid phase peptide synthesis or by using the FMOC strategy on aRaMPS multiple peptide synthesis system (DuPont Co., Wilmington Del.) asdescribed in Caprino and Han (J Org Chem 37:3404, 1972 which isincorporated by reference).

After a suitable peptide has been made, the peptide can be prepared in apharmaceutically acceptable composition that is capable of deliveringthe peptide into to a cell. Any known and available means can be usedfor delivering the peptide into a cell. For example, the peptide may beincorporated with a carrier moiety such as a liposome that is capable ofdelivering the peptide into the cytosol of a cell. Such methods are wellknown in the art (for example see Amselem et al., Chem Phys Lipids64:219-237, 1993 which is incorporated by reference). Alternatively, thepeptide can be modified to include specific transit peptides that arecapable of delivering the peptide into the cytoplasm of a cell or thepeptide can be delivered directly into a cell by microinjection ordelivery can be by intravenous administration in the intact individualin a pharmaceutically acceptable composition.

An effective amount of the peptide must be introduced into the cell sothat binding to the 14-3-3 protein takes place. It is to be understoodthat the amount of peptide necessary to be introduced into anyparticular cell is dependent upon the cell, but can be determined usingstandard dose/response analysis.

Based on the structural features of the critical amino acid sequence ofthe peptides of the present invention that permit the binding of thepeptide with the 14-3-3 protein, one can develop non-peptide derivativesthat are capable of binding to the 14-3-3 protein. It is believed thatat a minimum, non-peptide compositions that would be capable of bindingto a 14-3-3 protein would contain a phosphorylated serine-like structureand would be capable of being introduced into a cell.

The techniques for development of peptide mimetics are well known in theart. (See for example, Navia and Peattie, Trends Pharm Sci 14:189-195,1993; Olson et al., J Med Chem 36:3039-3049, 1993 which are incorporatedby reference). Typically this involves identification andcharacterization of the protein target as well as the protein ligandusing X-ray crystallography and nuclear magnetic resonance technology.In the case of 14-3-3 proteins, the complete sequence and the X-raycrystal structure for these proteins are known. Using informationlearned from the structure of the target protein and ligand peptide, apharmacophore hypothesis is developed and compounds are made and testedin an assay system. An assay system based upon displacement of ligandfrom 14-3-3 protein can be used.

Thus, in another embodiment the peptide ligands of this invention areused to detect non-peptide compositions capable of binding to 14-3-3peptide. A standard radioligand assay system can be used. (For example,see Bylund and Toews, Am J Physiol 265:L421-429, 1993 which isincorporated by reference). The method involves forming a mixture of alabeled peptide containing a sequence obtained from a Raf-1 sequencesurrounding serine-259 or serine-621, a protein comprising at least aportion of a 14-3-3 protein capable of binding to the labeled peptidewherein the 14-3-3 protein is immobilized on a solid substrate, and acandidate compound. The labeled protein contains at least the 6 aminoacids of SEQ ID NO: 1 and preferably about 15 amino acids in length. Thelabeled protein can also be the full length Raf-1 molecule or anyportion thereof so long as the portion binds to the 14-3-3 protein.

The labeled protein can comprise any of a variety of labels known in theart. Typically a radiolabeled protein is used. The radiolabeled peptidecan be prepared by attaching a radiolabeled group to the protein such asa phosphate group containing radiolabeled phosphorus or incorporationinto the protein structure such as with a methionine residue containingradiolabeled sulfur. One radiolabeling method is illustrated in theexamples below wherein the peptide is radiolabeled by phosphorylatingthe peptide using protein kinase A and [³²P]-γ-ATP. Radiolabeling canalso be accomplished with a number of other known methods includingusing either ³H or ¹²⁵I or biotinylation according to standard methods.For example, the Bolton Hunter Reagent can be used (ICN Chemicals,Radioisotope Division, Irvine, Calif.).

The composition of 14-3-3 to which the radioligand binds can be preparedby expression as a Glutathione-S-transferase 14-3-3 fusion protein inbacteria as discussed below. A standard ELISA-style plate assay can beused to bind radiolabeled ligand and then recover and measure the amountof bound ligand. (For example see Slack et al. BioTechniques7:1132-1138, 1989; Dower et al, J Immunol 142:4314-4320, 1989 which areincorporated by reference). Competitive inhibition of the binding of theradiolabeled peptide ligand to the 14-3-3 protein on addition of a testcompound can be evaluated by standard methods of analysis. (For example,see Rovati, Pharmacol Res 28:277-299, 1993 which is incorporated byreference). Alternatively, a non-radiolabeling method can be used todetect competitive displacement of the peptide ligand from the 14-3-3protein. Such non-radiolabeling methods can utilize labels includingenzymes, fluorescers, chemiluminescers, enzyme substrates or co-factors,enzyme inhibitors, particles, dyes and the like. Moreover, the assayscan also be performed by using solid phase peptide and labeled forms ofpurified, recombinant 14-3-3 protein.

The peptides of the present invention are useful in blocking theactivation of Raf-1 as a signal transducing peptide in response tocellular stimulation by insulin or a growth factor or in response to anoncogene product produced within the cell such as mutant Ras protein.Such blocking of signal transduction is likely to be particularlyeffective in treating tumors involving a mutant Ras protein or involvingoncogenic activation which utilizes a Ras protein pathway. Therequirement of Raf-1 activity for Ras effector signalling allows thepeptides of the present invention to interrupt the Ras protein pathwayof oncogenic activation in tumor cells. (see Prendergast et al.,BioEssays 16:187-191, 1994 which is incorporated by reference).

Peptides and derivatives thereof prepared in accordance with the presentinvention can be used to inhibit the intracellular activation of Raf-1in a mammalian cell and thereby provide a useful therapeutic compositionfor use in the treatment of diseases. Such diseases that can beadvantageously treated have as a component of the disease an increasedor inappropriate signal transduction mediated by Raf-1 such as, forexample, in the treatment of cancer. In such treatments, it can besometimes advantageous to target the peptides or derivatives of thepresent invention to the cancerous cells. Such specific targeting iswell known within the art of cancer treatment and the preparation ofsuitable formulations and methods requires no more than routineexperimentation.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

EXAMPLE 1

This example illustrates the direct binding of 14-3-3 zeta to a serinephosphorylated Raf-1 peptide.

A peptide containing a Raf-1 sequence surrounding serine-259 of Raf-1(residues 251-265, LSQRQRSTSTPNVHM, SEQ ID NO: 4, designated Raf-259)was synthesized, purified, and analyzed according to methods previouslydescribed (Lorenz et al., 1988 which is incorporated by reference).Synthesis according to these methods was on either an ABI model 432A(Perkin/Elmer, Foster City, Calif.) or a Symphony/Multiplex synthesizer(Rainin Instrument Co, Woburn, Mass.) using standard FMOC chemistry.Reagents for peptide synthesis were purchased from ABI, Rainin, andAdvanced ChemTech (Louisville, Ky.). Reagents for peptide synthesis werepurchased from ABI, Rainin, and Advanced ChemTech (Louisville, Ky.). Thepeptide was purified by C₁₈ reverse phase HPLC and their identifyconfirmed and their concentration determined by amino acid analysis(Beckman Model 6300, Beckman Instruments, Palo Alto, Calif.). Peptidesprepared by this method were shown to consist of a single species of thecorrect molecular weight by mass spectrometry (Washington UniversityMass Spectrometry Facility).

The 14-3-3 protein isoforms for use in binding studies with the abovepeptides were prepared as proteins fused with glutathione-S-transferase(GST) and immobilized to glutathione agarose beads. GST/14-3-3 fusionproteins were made by generating BamHI and EcoRI restrictionendonuclease sites (underlined) at the 5′ and 3′ ends respectively ofthe 14-3-3 cDNAs by polymerase chain reaction (PCR) using the followingisoform sequence specific pairs of primers: 14-3-3 beta,GAGGATCCACAATGGATAAAAGTGAG (SEQ ID NO: 11), GAGAATTCTTAGTTCTCTCCCTCCCCA(SEQ ID NO: 12); 14-3-3 eta GAGGATCCGGGGACCGGGAGCAGCTG (SEQ ID NO: 13),GAGAATTCTCAGTTGCCTTCTCCTGC (SEQ ID NO: 14); 14-3-3 tauGAGGATCCGAGAAGACTGAGCTGATC (SEQ ID NO: 15), GAGAATTCTTAGTTTTCAGCCCCTTC(SEQ ID NO: 16); 14-3-3 zeta ATTGGATCCGATAAAAATGAGCTGGTTC (SEQ ID NO:17), TTGAATTCAATTTTCCCCTCCTTCTCCT (SEQ ID NO: 18). The cDNA productswere digested with BamHI and EcoRI, subcloned into the vector pGEX-KT(Guan and Dixon, Anal Biochem 192: 262-7, 1991 which is incorporated byreference). The vector was used to transform the DH5α strain of E. Coli.GST-14-3-3 fusion protein expression in log-growth phase bacterialcultures was accomplished by induction with 100 μM isopropylβ-D-thiogalactopyranoside (IPTG; Gold Biotechnology, St. Louis) for 4hours. Cells were disrupted by sonication and lysates, containing 1%Triton X-100, were incubated with glutathione agarose beads (Sigma, St.Louis) at 4° C. overnight. Fusion proteins were eluted by incubationwith 10 mM reduced glutathione (Sigma, St. Louis) in phosphate bufferedsaline for 1 hour at room temperature, dialyzed in phosphate bufferedsaline and quantitated by Coomassie staining SDS polyacrylamine gelscompared against bovine serum albumin standards.

The Raf-259 peptide (SEQ ID NO: 4) was phosphorylated in vitro usingprotein kinase A (PKA) and [³²P]-γ-ATP following the manufacturersinstruction (PKA, New England Biolabs) with 20 μCi of [³²P]-γ-ATP(NEN-Dupont, 6000 Ci/mmol). The efficiency of phosphorylation wascalculated to be between 7 and 12% based upon the specific activity ofthe labelled peptide.

The labeled peptide was then incubated with immobilized 14-3-3 zetafusion protein and binding of the labelled peptide was measured. Theresults shown in FIG. 1 demonstrated that the labeled phosphorylatedpeptide bound to the 14-3-3 zeta coupled beads, but not to theglutathione beads alone. Binding was specific for phosphoserine becauseit could be displaced with phosphorylated peptide, but not with theunphosphorylated peptide. Binding was also sequence specific. Neitherfree phosphoserine nor a threonine phosphorylated casein kinase IIsubstrate peptide (Arg—Arg—Arg—Glu—Glu—Glu-Xaa-Glu—Glu—Glu where Xaa isphosphorylated tyrosine, SEQ ID NO: 19) displaced the boundphosphorylated peptide nor did a tyrosine phosphorylated peptide(Xaa-Glu—Glu-Ile-Gln-Pro-Ala-Lys where Xaa is a phosphorylated tyrosine,SEQ ID NO: 20). The interaction of the phosphorylated peptide with14-3-3 protein was also of high affinity having an IC₅₀ of approximately1-2 μM for the phosphorylated peptide (FIG. 2). Because the calculatedefficiency of in vitro phosphorylation of the peptide ranged between7-12%, the true IC₅₀ was approximately 70-240 nM.

Surface plasmon resonance (SPR) was used to directly measure theaffinity of the interaction of 14-3-3 zeta for the Raf-1 peptide. Theseexperiments were performed using the BIAcore 2000 (Pharmacia). The basicprinciples and detection methods have been reviewed previously (Jonssonet al. Biotech. 11:620-7, 1991; Jonsson et al. Ann Bio Clin 51:19-26,1993 which are incorporated by reference). In brief, 5 μl of thebiotinylated raf-259 peptide (1 nM) was immobilized ontostreptavidin-coated (SA5, Pharmacia) sensor chips at a flow rate of 5μl/min at 25° C. This generally resulted in an RU value of 20-25 units.To phosphorylate the immobilized peptides, 5 U of PKA in a buffercontaining 200 μM ATP in 50 μl was infused over the chip at a flow rateof 2 μl/min at 30° C.

When the unphosphorylated Raf-1 peptide (SEQ ID NO: 4) was attached tosensor surface and tested for its ability to bind injected 14-3-3 zeta,no significant binding was observed (FIG. 3). Following in situphosphorylation of the peptide and re-injection of 14-3-3 zeta over thesensor surface, binding was clearly demonstrated (FIG. 3). This bindingwas specific to the 14-3-3 moiety as GST alone did not bind to eitherthe unphosphorylated or phosphorylated peptide (data not shown).

Because the surface plasmon resonance detects binding in real time, thismethod can be used to directly determine the rates of association(k_(on)) and dissociation (k_(off)). For these kinetic measurements,GST/14-3-3 fusion proteins were injected over a range of concentrationsbetween 200 nM and 5 μM. The “on” rate, k_(on), was determined bycalculating dR/dt for the initial linear portion of association usingthe Biaevaluation software (Pharmacia). Because ligand rebinding to thesensor chip can affect the “off” rate (k_(off), Panayotou et al. MolCell Biol 13:3567-76, 1993 which is incorporated by reference), wemeasured the “off” rate in the presence of 50 μM free ligand by usingthe coinject function of the Biacore. The “off” rate measured in theabsence of 50 μM free ligand was approximately 10-fold lower.

The biotinylated, unphosphorylated Raf-1 peptide (SEQ ID NO: 4) wascoupled to avidin coated chip surface to study the kinetics of itsbinding to 14-3-3 zeta. The association rate was determined to beapproximately 2.3×10⁴ M⁻¹×s⁻¹ and the dissociation rate was determinedto be approximately 2.8×10⁻³ s⁻¹. To minimize the effects of proteinrebinding to the surface of the sensor chip, the dissociation rate wasmeasured in the presence of 50 μM free phosphorylated Raf-1 peptide.Given that the equilibrium dissociation constant, K_(D) can bedetermined by K_(D)=k_(off)/k_(on), the apparent K_(D) measured by SPRis approximately 122 nM. These results show that 14-3-3 binds to thephosphoserine peptides of the present invention with high affinity.

EXAMPLE 2

This example illustrates the sequence specificity for the binding of thephosphoserine Raf-1 peptides to 14-3-3 proteins.

As the Raf-1 peptide used above (SEQ ID NO: 4) contains potential PKAphosphorylation sites at position 257 and 259, confirmation thatphosphoserine-259 is the critical phosphorylated residue was required.Raf-259 peptides were, therefore, synthesized with phosphoserines atpositions corresponding to position 257 (pS-Raf-257) (SEQ ID NO: 21),259 (pS-raf-259)(SEQ ID NO: 5) or both 257 and 259 (pS-Raf-257, 259)(SEQID NO: 22). The peptides were synthesized according to the methods inExample 1 with the exception that the phosphoserine containing peptideswere synthesized using FMOC-Ser(PO(OH,OBzl))—OH (NOVAbiochem, San Diego,Calif.) following the manufacturer's recommendations. Each of thepeptides was purified by HPLC and the integrity of each peptide wasconfirmed by mass spectroscopy. The peptides were based upon sequencesin Raf-1 surrounding serine-259 and serine 621 and are as shown in Table1.

TABLE 1 Sequence Name SEQ ID NO: L S Q R Q R S T S  T P N V H M Raf-2594 L S Q R Q R pS  T S  T P N V H A pS-Raf-257 21 L S Q R Q R S T pS T PN V H A pS-Raf-259 5 L S Q R Q R   pS T pS T P N V H A pS-Raf-257, 25922 L S Q A Q A S T pS T P N V H A 254/256RA 26 L S Q A Q R S T pS T P NV H A 254RA 27 L S Q R Q A S T pS T P N V H A 256RA 28 L S Q A Q S R TpS T P N V H A R-2 29 L S Q A R Q S T pS T P N V H A R-4 30 L S Q R Q RS T pS T A N V H M PA 31 L P K I N R S A pS E P S L H R pS-Raf-621 10

The ability of each peptide to inhibit binding of 14-3-3 zeta to thephosphorylated peptide was then tested in a range of concentrationsbetween 1 and 50 μM. 14-3-3 zeta was prepared according to the methodsin Example 1 and binding affinities and specificities were determinedusing the peptides in Table 2. The 14-3-3 zeta fusion proteins (0.5-1.0μM) were pre-incubated with a given peptide at concentrations between 1μM and 50 μM. 14-3-3 zeta was then infused across a sensor chip surfacewhich had been previously coupled with the phosphorylated Raf-259peptide (SEQ ID NO: 4). IC₅₀ values were determined by plotting theconcentration of competitor peptide required to achieve a 50% reductionin equilibrium binding. Because concentrations of 14-3-3 were required(500-1000 nM) to reach equilibrium binding that were much higher thanthe apparent K_(D) (100-150 nM), the IC₅₀ values that were derivedprovide only a relative affinity for the different peptides used (Payneet al., Proc Natl Acad Sci USA 90:4902-4906, 1993 which is incorporatedby reference).

IC₅₀ values for the interaction of each of the labeled proteins with14-3-3 zeta is shown in Table 2.

TABLE 2 SEQ SEQ ID ID Peptide NO IC50(μM) Peptide NO IC50(μM) pS-Raf-2595 6 256RA 28 19 pS-Raf-257 21 >50 R-2 29 >50 pS-Raf-257,259 22 >50 R-430 11 254/256RA 26 >50 PA 31 25 254RA 27 9 pS-Raf-621 10 1

The pS-raf-259 peptide (SEQ ID NO: 5) was found to compete with an IC₅₀of approximately 6 μM (Tables 3 and 4). In contrast, the pS-raf-257 (SEQID NO: 21) peptide and the pS-raf-257,259 peptide (SEQ ID NO: 22)demonstrated little, if any, inhibition at the highest concentrationtested, 50 μM. This demonstrates that phosphorylation of serine-259 iscritical for 14-3-3 zeta binding. Furthermore, the inability of thepS-raf-257-259 (SEQ ID NO: 22) peptide to bind suggests that the serineat position 257 is also important. As the only difference betweenpS-raf-257 (SEQ ID NO: 21) and pS-raf-259 (SEQ ID NO: 5) peptides is theposition of the phosphoserine, high affinity binding of 14-3-3 zeta toserine phosphate is exquisitely sequence specific.

To further define the 6 amino acid binding motif (SEQ ID NO: 1),sequences similar to the amino acids surrounding serine-259 in otherproteins known to bind 14-3-3 were identified (Table 3). Interestingly,CDC25 shares the motif Arg-Xaa₁-Arg-Ser-Xaa₂-Ser-Xaa₃-Pro (where Xaa₁,Xaa₂ and Xaa₃ are any amino acid) (SEQ ID NO: 23) while polyoma middle Tantigen contains a slightly shorter version of the motif(Arg-Ser-Xaa₁-Ser-Xaa₂-Pro) (SEQ ID NO: 24). Surprisingly, Raf-1contains both instances of the motif, at positions 256-261 and 618-623respectively; both are known to be phosphorylation sites in vivo(Morrison et al., supra). This suggested that the binding motif isrelated to the sequence Arg-Xaa₁-Arg-Ser-Xaa₂-Xaa₃-Xaa₄-Pro (where Xaa₁and Xaa₂ are any amino acid, Xaa₃ is phosphoserine and Xaa₄ is any aminoacid) (SEQ ID NO: 25).

TABLE 3 SUBSTANCES KNOWN TO BIND 14-3-3 SUBSTANCE MOTIF SEQ ID NO Raf-1(254-261) RSTSTP 39 Raf-1 (616-623) RSASEP 40 B-RAF (241-248) RSSSAP 41B-RAF (605-621) RSASEP 40 Polyoma Middle T (268-275) RSHSYP 42 CDC25B(301-308) RSPSMP 43 PKC-epsilon RSKSAP 44 PKC-gamma RSPSSP 45 BCR(368-373) RSQSQN 46 tyrosine hydroxylase RHASSP 47

TABLE 4 PUTATIVE PHOSPHORYLATED LIGANDS FOR 14-3-3 SUBSTANCE MOTIF SEQID NO A-RAF (206-213) RSTpSTP 39 A-RAF (574-581) RSApSEP 40 Bad RSRpSAP48 Mos RSCpSIP 49 CDC25C (208-215) RSPpSMP 43 Glucocorticoid ReceptorRSTpSRP 50 KSR-1 RSPpSFP 51 PLC-gamma RSEpSSP 52 p85 subunit of PI-3kinase RSPpSIP 53 PTP-MEG RSPpSKP 54 PTP-epsilon RSPpSGP 55 PTP-muRSVpSSP 56 SNF1 RSRpSYP 57 5′AMP Kinase RSQpSKP 58

The importance of the arginine residues in defining the 14-3-3 zetabinding motif was tested by synthesizing phosphopeptides with alaninessubstituted for either or both arginines (254/256RA, SEQ ID NO: 26;254RA, SEQ ID NO: 27; and 256RA, SEQ ID NO: 28 according to Table 1). Asshown in Table 2, the peptide with alanine substituted for botharginines (254/256RA, SEQ ID NO: 26) exhibited no detectable inhibitionat concentrations up to 50 μM demonstrating that one or both arginineresidues are critical for binding. The 254RA (SEQ ID NO: 27) peptidecompeted almost as efficiently as wild-type (9 μM) while the 256RApeptide (SEQ ID NO: 28) competed much less efficiently. Although thisdemonstrated that the arginine in position −3 from the phosphoserine iscritical for binding, the inability of the alanine substitution at −5 tocompletely abrogate binding suggested that basic residues in otherpositions might also contribute to the binding affinity. The relativeaffinities of two additional peptides with the arginine placed in the −2or −4 positions were therefore analyzed. The peptide with arginine inthe −2 position (R−2, SEQ ID NO: 29) did not compete for binding at thehighest concentration tested (50 μM) confirming that the position of thearginine residue is clearly important. However, the peptide witharginine in the −4 position (R−4, SEQ ID NO: 30) competed almost as wellas the peptide with arginine in the −3 position (254RA, SEQ ID NO: 28).We concluded that an arginine residue is required in the −3 and/or −4position from the phosphorylated serine.

To determine the importance of the proline residue, anotherphosphorylated peptide with alanine substituted for proline (PA) wasgenerated (SEQ ID NO: 31). The PA peptide demonstrated an IC₅₀ ofapproximately 25 μM confirming that the proline residue is important butalso suggesting that this position may tolerate other residues (Table2).

To determine whether other predicted sequences containing the motifcould bind 14-3-3, we tested a phosphopeptide corresponding to Raf-1residues 613-627 (pS-raf-621, SEQ ID NO: 10). The IC₅₀ of this peptidewas approximately 1 μM. The ability of the phosphorylated raf-621peptide to bind at high affinity confirmed that the motif can be used topredict other proteins that bind 14-3-3. Its affinity, which is sixtimes higher than that of the raf-259 peptide (SEQ ID NO: 5) alsoindicated that ser-621 of Raf-1 is the preferred binding site for 14-3-3in contrast to what has been previously reported (Michaud et al.,supra). As this latter peptide contains only one of the two N-terminalarginine residues, the minimal binding sequence for 14-3-3 zeta isArg-Ser-Xaa₁-Xaa₂-Xaa₃-Pro where Xaa₁ is any amino acid, Xaa₂ isphosphoserine, and Xaa₃ is any amino acid (SEQ ID NO: 1).

Comparison of proteins known to bind 14-3-3 demonstrated that manycontain sequences similar to those surrounding serine-259 of Raf-1(Table 1). This analysis allowed us to deduce a putative motif for14-3-3 binding as being Arg-Ser-Xaa₁-Ser-Xaa₂-Pro where Xaa₁ and Xaa₂are any amino acid (SEQ ID NO: 1). The integrity of this derived motifwas tested by using a series of alanine substituted phosphorylatedpeptides, a panel of degenerate peptides, as well as by testing whether14-3-3 could bind to a peptide from a predicted site. Thecarboxy-terminal proline and the amino-terminal arginine residues wereimportant for binding. Although analysis of the position dependence ofthe arginine demonstrated high affinity binding in either the −3 and −4positions, the −3 position is required for phosphorylation by proteinkinases. We believe, therefore, that arginine in the −3 position is thecritical determinant for 14-3-3 binding in vivo.

The inability of the doubly phosphorylated peptide, pS-Raf-257,259 (SEQID NO: 22), to bind 14-3-3 suggested that the serine residue in the −2position is also important. Experiments with degenerate peptidesconfirmed that specificity is conferred by residues in the −2 as well asthe −3 and +2 positions relative to the phosphoserine. It is possiblethat other residues can substitute for the arginine, serine and prolineinasmuch as other proteins known to bind 14-3-3 like bcr and tyrosinehydroxylase contain related sequences (Table 3) but do not contain theexact motif. An improved definition of the motif will be critical in theidentification of other novel phosphoserine containing peptides basedupon proteins that putatively bind 14-3-3 (represented in Table 4 inphosphorylated form).

Our data suggests that other proteins containing the motif, ifappropriately phosphorylated, might bind 14-3-3. The SWISS-PROT proteindatabase was, therefore, searched for eukaryotic proteins that containthe sequence Arg-Ser-Xaa₁-Ser-Xaa₂-Pro where Xaa₁ and Xaa₂ are any aminoacid (SEQ ID NO: 32). As expected, c-Raf-1, CDC25 and polyoma middle Tantigen were identified. Kinases closely related to c-Raf-1 likeA-Raf-1, B-Raf-1, mos, mil and MEKK also contain the motif. Someadditional proteins that contain the motif are shown in Table 4.

Based upon these results, the inventors herein believe that the primary14-3-3 binding site on Raf-1 may not be Ser-259 as proposed previously(Michaud et al, supra), but rather Ser-621. Ser-259 phosphorylation isinduced only after growth factor stimulation but 14-3-3 is boundconstitutively to Raf-1 in unstimulated cells (Li et al. supra; Dent etal., Science 268:1902-6, 1995 which are incorporated by reference).Therefore, it is not likely that Ser-259 is the primary binding site ofRaf-1 in vivo. Ser-621, a Raf 1 site which is constitutivelyphosphorylated (Morrison et al., supra), is, therefore, the preferred14-3-3 binding site. Michaud et al. suggested serine-259 as the majorbinding site, because baculovirus expressed protein is aberrantlyphosphorylated at both serine-259 and serine-621 (Morrison et al.,supra).

Binding of 14-3-3 may not directly activate Raf-1 per se as Raf-1/14-3-3complexes are present in unstimulated cells (Li et al., supra). Butmutation of serine-621 in Raf-1 inhibits the association of 14-3-3(Michaud et al., supra) and renders the kinase inactive. 14-3-3 bindingto serine-621 is, therefore, believed to be required for Raf-1 kinaseactivity. Although the inventors herein do not intend that thisinvention be limited in any way by a particular mechanism of action, onepossible explanation for the results reported herein is that 14-3-3functions as a chaperone, to promote or stabilize an activatableconformation of Raf-1. This would explain why disruption of the14-3-3/Raf-1 complexes with the phosphorylated peptide blocks Raf-1activation. 14-3-3 interactions with phosphorylated serine-259 aftergrowth factor treatment may also be important as serine-259 is requiredfor PKC activation of Raf-1 (Kolch et al., Nature 364:249-252, 1993which is incorporated by reference).

EXAMPLE 3

This example confirms the critical motif residues required for bindingto 14-3-3 protein using degenerate peptides based upon Serine-621 ofRaf-1.

A series of degenerate peptides based upon phosphoserine Raf-621 peptide(SEQ ID NO: 10) were synthesized to confirm that the arginine in the −3position, the serine in the −2 position and the proline in the +2position were the critical motif residues (Table 5). Six pools ofpeptides were synthesized with all twenty amino acids placed inpositions −5, −3, −2, −1, +1 and +2 respectively. Each pool of twentypeptides was tested as competitive inhibitors for binding. We reasonedthat if a particular position is not critical for binding, the pool oftwenty peptides should compete as well as wild-type peptide for binding.On the other hand, if a particular amino acid is required at aparticular position, only a small fraction of the pool will be able tocompete for binding, thus increasing (up to 20 fold) the concentrationof peptide needed to inhibit binding. Pools with degenerate amino acidsin positions −5, −1 and +1 competed as well as wild-type demonstratingthat these position are not critical for binding. As expected, poolswith degenerate amino acids in position −3, −2 and +2 were weakerinhibitors of binding confirming that these are the critical positions.

TABLE 5 PEPTIDE IC50 (μM) Isoform PA RA pS-Raf-259 pS-Raf-257,259pS-Raf-257 pS-Raf-621 zeta 25 >50 6 >50 >50 1 eta 20 >50 5 >50 >50 3beta 24 >50 6 >50 >50 2 tau 27 >50 5 >50 >50 2

EXAMPLE 4

This example demonstrates that the phosphoserine Raf-1 peptides bind todifferent isoforms of 14-3-3 with the same affinity and specificity.

There are seven known isoforms of 14-3-3 which form homo- andheterodimers in vivo. If different isoforms of 14-3-3 could bind toproteins with distinct specificities, heterodimeric forms of 14-3-3might function as modular linker proteins, bringing together differentproteins into a single complex. We were, therefore, interested indetermining whether different 14-3-3 isoforms recognize phosphoserine ina sequence specific fashion. This hypothesis was tested by expressingand purifying three other 14-3-3 isoforms, eta, beta and tau, andmeasuring their affinity towards the panel of phosphorylated peptidesgenerated above. Using the Surface Plasmon Resonance method, the “on”and “off” rates for 14-3-3 binding to the phosphorylated Raf-259 peptidewere calculated (Table 6). All four isoforms demonstrated very similarapparent binding affinities.

TABLE 6 Isoform k_(on)(10⁴M⁻¹s⁻¹) k_(off)(s⁻¹) K_(D)(nM) zeta 2.300.0028 122 eta 2.26 0.0029 128 beta 2.20 0.0032 145 tau 2.50 0.0036 144

Competitive inhibition with the mutated phosphorylated Raf-1 peptideswas used to determine whether their specificities were similar (Table7). In each case, the calculated IC₅₀s were very similar to thosecalculated for 14-3-3 zeta. All four 14-3-3 isoforms exhibited little tono affinity towards the RA peptide and intermediate affinities towardsthe PA peptide.

TABLE 7 % BINDING COMPETITOR PEPTIDE SEQ ID @ 10 μM No peptide 100% pS-Raf-621 L P K I N R S A pS E P S L H R 10 <5% −5 L P K X N R S A pS EP S L H R 33 <5% −3 L P K X N X S A pS E P S L H R 34 17% −2 L P K I N RX A pS E P S L H R 35 27% +1 L P K I N R S X pS E P S L H R 36 <5% +1 LP K I N R S A pS X S L H R 37 <5% +2 L P K I N R S A pS E P S L H R 38<56% 

These results suggest that the binding affinity and sequence specificityof multiple 14-3-3 isoforms for serine phosphorylated peptides are verysimilar suggesting that the peptides disclosed herein will inhibit thesignalling functions of all isoforms of 14-3-3.

EXAMPLE 5

This example demonstrates that 14-3-3 protein can blockdephosphorylation of Raf-1.

To confirm that 14-3-3 physically contacts the phosphoserine residue, wetested whether addition of 14-3-3 could block the ability of theserine/threonine phosphatase, PP1, to dephosphorylate Raf-1. One μg ofGST-raf fusion protein adsorbed to glutathione agarose beads wasphosphorylated in vitro with purified bovine PKA (Sigma) and[³²P]-γ-ATP. Ten μg of purified GST-14-3-3 β or a GST-P56-lck SH2 (SantaCruz Biotechnology, Santa Cruz, Calif.) domain was added to thephosphorylated Raf fusion protein in PBS and incubated at roomtemperature for 1 hour. The beads were collected by centrifugation,resuspended in 25 μl buffer containing 50 mM Tris-HCl (pH 7), 0.1 mMEDTA, 5 mM DTT, 0.2 mM MnCl₂, 200 mg/ml bovine serum albumin and 1 U ofrecombinant protein phosphatase 1 (New England BioLabs, Beverly, Mass.)and incubated for 1 hour at 37° C. Proteins were analyzed by PAGE andquantitated using a phosphorimager (Molecular Dynamics, Sunnyvale,Calif.).

Incubation of phosphorylated Raf-1 with 14-3-3 completely blocked theability of PP1 to dephosphorylate Raf-1 (FIG. 4). This was specific to14-3-3 because addition of a control fusion protein, GST-p56-lck SH2,had no effect on the ability of PP1 to dephosphorylate Raf-1.

EXAMPLE 6

This example demonstrates that the Raf-1 phosphoserine peptides inhibitthe 14-3-3/Raf-1 interaction in vitro.

The ability of phosphorylated peptides of the present invention to block14-3-3/Raf-1 complex formation was tested as follows.

NIH 3T3 cells and T cell hybridoma (DO11.10) were washed twice in coldphosphate-buffered saline, lysed in cold NP40 lysis buffer (Muslin etal., Mol Cell Biol 13:4197-202, 1993 which is incorporated by reference)and cleared by centrifugation.

The lysates prepared from NIH 3T3 cells were incubated for 1 hr at 4° C.with immobilized 14-3-3 in the presence or absence of phosphorylated andunphosphorylated Raf-1 peptides (SEQ ID NO: 5 and SEQ ID NO: 4,respectively). After washing three times in lysis buffer, Raf-1 bindingwas analyzed by immunoblotting using a polyclonal anti-Raf-1 antibody(Santa Cruz Biotechnology). In lane 1 lysate was added to immobilized14-3-3 in the absence of added peptide. In lane 2 lysate was added toGST alone immobilized on glutathione-agarose. In lane 3 lysate was addedto immobilized 14-3-3 in the presence of unphosphorylated Raf-259peptide (SEQ ID NO: 4, 10 μM). In lane 4 lysate was added to immobilized14-3-3 in the presence of phosphorylated pS-Raf-259 peptide (SEQ ID NO:5, 10 μM).

Incubation of 14-3-3 zeta with the phosphorylated raf-259 peptide(pS-raf-259)(SEQ ID NO: 5) completely inhibited its ability to bindRaf-1 (FIG. 5, lane 4). In contrast, incubation of cell lysates with theunphosphorylated Raf-1 peptide (SEQ ID NO: 4) had no effect on theability of 14-3-3 zeta to bind to full-length Raf-1 (FIG. 5, lanes 3).

The phosphopeptide could also promote the disassociation of preformed14-3-3/Raf-1 complexes (FIG. 6). Either unphosphorylated orphosphorylated Raf-259 peptides (SEQ ID NO: 4 and SEQ ID NO: 5,respectively) were incubated with cell lysates prepared from a mouse Tcell hybridoma for 1 hr at 4° C. Raf-1 immunoprecipitates were washed 3times in lysis buffer and separated by SDS-PAGE. Associated 14-3-3protein was detected by immunoblotting using a rabbit polyclonalanti-14-3-3 beta antibody (Santa Cruz Biotechnology; ab in FIG. 6). Inlane 1 unphosphorylated Raf-259 peptide (SEQ ID NO: 4, 100 μM) was addedto cell lysate. In lane 2 phosphorylated pS-Raf-259 peptide (SEQ ID NO:5, 10 μM) was added to cell lysate. In lane 3 phosphorylated pS-Raf-259peptide (SEQ ID NO: 5, 100 μM) was added to cell lysate.

Addition of the pS-Raf-259 peptide (SEQ ID NO: 5) at either 10 μM or 100μM concentrations disassociated Raf-1/14-3-3 complexes (FIG. 6, lanes 2and 3). Incubation of cell lysates with the unphosphorylated Raf-259peptide (SEQ ID NO: 4) had no effect on Raf-1/14-3-3 complexes (FIG. 6,lane 1). The 14-3-3/Raf-1 complexes can, therefore, be disassociated byincubation with the phosphorylated Raf-1 peptide (SEQ ID NO: 5).

EXAMPLE 7

This example demonstrates that the Raf-1 phosphoserine peptides inhibitof 14-3-3/Raf-1 interaction in vivo in a model of oocyte maturation.

Phosphorylated Raf-1 peptides were microinjected into Xenopus oocytes.Because microinjection of 14-3-3 protein into frog oocytes activatesRaf-1 inducing oocyte maturation (Fantl et al., supra), we reasoned thatinhibition of 14-3-3 interactions using the phosphorylated Raf-1peptides should block Raf-1 activation and subsequent oocyte maturation.Insulin was used to stimulate oocyte maturation and maturation wasmeasured by assessing germinal vesicle breakdown (GVBD, Fabian et al., JCell Biol 122:645-52, 1993; Muslin et al., supra which are incorporatedby reference).

Large oocytes (Dumont stage VI) were removed from adult female frogs.Oocytes were manually dissected and collagenase treated. Oocytes weremaintained in 1× modified Barth's saline with added bovine serumalbumin, Ficoll 400, and antibiotics as described (Muslin et al.,supra). Each oocyte was injected with 50 nmoles of peptide or withwater. The final concentration of the peptide was estimated as 50 μM andthe half-life measured as 15-20 minutes. Oocytes were treated withinsulin (8.25 μg/ml) and incubated for 24 hours at 18° C.

In the presence of insulin, microinjection of the phosphorylated Raf-259peptide (SEQ ID NO: 5) but not the unphosphorylated Raf-259 peptide (SEQID NO: 4) inhibited insulin stimulated GVBD (FIG. 7). Consistent withthis result, injection of the pS-Raf-621 peptide (SEQ ID NO: 10)significantly inhibited insulin stimulated GVBD but the RA peptide (SEQID NO: 26) did not (data not shown). These results demonstrate thatphosphoserine Raf-1 peptides can inhibit oocyte maturation.

The mechanism of inhibition of oocyte maturation by the phosphoserineRaf-1 peptides was further elucidated by determining the effect of thephosphoserine-containing Raf-1 peptides on Raf-1 kinase activity in theinsulin-treated oocytes.

Immature Xenopus oocytes were injected with phosphorylated orunphosphorylated peptide as above. Oocytes were stimulated with insulinfor 24 hours and protein lysates were made. The oocytes were then lysedas described previously (Muslin et al., supra). Raf-1 immunoprecipitateswere washed and then incubated in kinase buffer in the presence of 100ng of recombinant polyhistidine-tagged MEK as a substrate as described(MacNicol et al., Mol Cell Biol 15: 6686-6693, 1995 which isincorporated by reference). The samples were then subjected to SDS-PAGEand the phosphorylated substrate was visualized by autoradiography.

With reference to FIG. 8, lane 1 shows unstimulated water-injectedoocytes; lane 2, insulin-stimulated water-injected oocytes; lane 3,insulin-stimulated oocytes injected with phosphorylated pS-Raf-259peptide (SEQ ID NO: 5); and lane 4, insulin-stimulated oocytes injectedwith unphosphorylated Raf-259 (SEQ ID NO: 4).

Raf-immunoprecipitates from oocytes microinjected with thephosphorylated Raf-1 peptide, pS-Raf-259 (SEQ ID NO: 5), demonstratedlittle to no activation of Raf-1 kinase as compared with the oocytesmock-injected or injected with the unphosphorylated Raf-1 peptide (SEQID NO: 4) after insulin treatment. These results are consistent withprevious work demonstrating that 14-3-3 is required for Raf-1 activation(Irie et al., Science 265:1716-1719, 1994; Freed et al., Science265:1713-6, 1994; and Li et al., supra which are incorporated byreference).

EXAMPLE 8

This example illustrates the effectiveness of the administration of thephosphoserine Raf-1 peptides in producing a reversion of oncogenicallytransformed cells.

Because Raf-1 is activated by Ras and is required for transformation byRas, the inventors herein believe that inhibition of Raf-1 function willblock the transforming effects mediated by a Ras protein. It isgenerally believed that intracellular Ras protein plays a major role inhuman carcinogenesis both as an oncogenic protein protein from a mutatedras gene and as a normal Ras protein mediating the effects of agrowth-factor oncogene (Prendergast et al. supra). Using standardmicroinjection techniques, phosphoserine Raf-259 and Raf-621 peptidesare injected into the cytoplasm of cells of oncogenically transformed ina manner mediated by a Ras protein and reversion is assessed bydetecting changes in the morphology of the transformed cells and byperforming cell cycle analysis according to methods well known in theart (Dobrowolski et al, Mol Cell Biol 14:5441-5449, 1994; Smith et al.,Nature 320:540-543, 1986; Kung et al, Exp Cell Res 162:363-371, 1986which are incorporated by reference). Briefly, these experiments areconducted as follows:

NIH 3T3 are transformed by incubating the cells with an oncogene such asthe membrane-associaed receptor-like proteins, src, fms and fes (Smithet al, supra). Alternatively, NIH 3T3 cells can be transformed bymicroinjection of an oncogenic Ras protein such as, for example, p21 Rasproteins (Kung et al, supra).

Transformed cells can be identified morphologically in that they take ona spindle-shaped appearance and in phase-contrast optics the cellsbecome darkened and surrounded by a bright refractile border. Inaddition, the ³H-thymidine incorporation of the transformed cells can bedetermined by standard methods.

The transformed cells prepared by one of the methods indicated above,are microinjected with a phosphoserine Raf-259 or Raf-621 peptide suchas, for example, SEQ ID NO: 10 or SEQ ID NO: 5. Reversion of the cellcan then be detected by monitoring cell morphology or ³H-thymidineincorporation. Alternatively, the peptide can be microinjected prior totransformation and the prevention of transformation monitored. As apositive control for reversion or prevention of transformation, thec-ras protein the monoclonal antibody, Y13-259, can be microinjectedinto the cells.

These experiments illustrate the effectiveness of the phosphoserine Rafpeptides of the present invention in treating cancer cells.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

58 6 amino acids amino acid linear peptide unknown Modified-site /note=“Any amino acid” Modified-site /note= “A phosphorylated serine”Modified-site /note= “Any amino acid” 1 Arg Ser Xaa Xaa Xaa Pro 1 5 8amino acids amino acid linear peptide unknown Modified-site /note= “Anyamino acid” Modified-site /note= “Any amino acid” Modified-site /note=“A phosphorylated serine” Modified-site /note= “Any amino acid” 2 ArgXaa Arg Ser Xaa Xaa Xaa Pro 1 5 9 amino acids amino acid linear peptideunknown Modified-site /note= “Any amino acid” Modified-site /note= “Anyamino acid” Modified-site /note= “Any amino acid” Modified-site /note=“A phosphorylated serine” Modified-site /note= “Any amino acid” 3 LysXaa Xaa Arg Ser Xaa Xaa Xaa Pro 1 5 15 amino acids amino acid linearpeptide unknown 4 Leu Ser Gln Arg Gln Arg Ser Thr Ser Thr Pro Asn ValHis Met 1 5 10 15 15 amino acids amino acid linear peptide unknownModified-site /note= “A phosphorylated serine” 5 Leu Ser Gln Arg Gln ArgSer Thr Xaa Thr Pro Asn Val His Ala 1 5 10 15 10 amino acids amino acidlinear peptide unknown Modified-site /note= “A phosphorylated threonine”6 Arg Arg Arg Glu Glu Glu Xaa Glu Glu Glu 1 5 10 8 amino acids aminoacid linear peptide unknown Modified-site /note= “A phosphorylatedtyrosine” 7 Xaa Glu Glu Ile Ser Pro Ala Lys 1 5 6 amino acids amino acidlinear peptide unknown 8 Arg Ser Thr Ser Thr Pro 1 5 6 amino acids aminoacid linear peptide unknown 9 Arg Ser Ala Ser Glu Pro 1 5 15 amino acidsamino acid linear peptide unknown Modified-site /note= “A phosphorylatedserine” 10 Leu Pro Lys Ile Asn Arg Ser Ala Xaa Glu Pro Ser Leu His Arg 15 10 15 26 base pairs nucleic acid single linear cDNA unknown 11GAGGATCCAC AATGGATAAA AGTGAG 26 27 base pairs nucleic acid single linearcDNA unknown 12 GAGAATTCTT AGTTCTCTCC CTCCCCA 27 26 base pairs nucleicacid single linear cDNA unknown 13 GAGGATCCGG GGACCGGGAG CAGCTG 26 26base pairs nucleic acid single linear cDNA unknown 14 GAGAATTCTCAGTTGCCTTC TCCTGC 26 26 base pairs nucleic acid single linear cDNAunknown 15 GAGGATCCGA GAAGACTGAG CTGATC 26 26 base pairs nucleic acidsingle linear cDNA unknown 16 GAGAATTCTT AGTTTTCAGC CCCTTC 26 28 basepairs nucleic acid single linear cDNA unknown 17 ATTGGATCCG ATAAAAATGAGCTGGTTC 28 28 base pairs nucleic acid single linear cDNA unknown 18TTGAATTCAA TTTTCCCCTC CTTCTCCT 28 10 amino acids amino acid linearpeptide unknown Modified-site /note= “A phosphorylated threonine” 19 ArgArg Arg Glu Glu Glu Xaa Glu Glu Glu 1 5 10 8 amino acids amino acidlinear peptide unknown Modified-site /note= “A phosphorylated tyrosine”20 Xaa Glu Glu Ile Gln Pro Ala Lys 1 5 15 amino acids amino acid linearpeptide unknown Modified-site /note= “A phosphorylated serine” 21 LeuSer Gln Arg Gln Arg Xaa Thr Ser Thr Pro Asn Val His Ala 1 5 10 15 15amino acids amino acid linear peptide unknown Modified-site /note= “Aphosphorylated serine” Modified-site /note= “A phosphorylated serine” 22Leu Ser Gln Arg Gln Arg Xaa Thr Xaa Thr Pro Asn Val His Ala 1 5 10 15 8amino acids amino acid linear peptide unknown Modified-site /note= “Anyamino acid” Modified-site /note= “Any amino acid” Modified-site /note=“Any amino acid” 23 Arg Xaa Arg Ser Xaa Ser Xaa Pro 1 5 6 amino acidsamino acid linear peptide unknown Modified-site /note= “Any amino acid”Modified-site /note= “Any amino acid” 24 Arg Ser Xaa Ser Xaa Pro 1 5 8amino acids amino acid linear peptide unknown Modified-site /note= “Anyamino acid” Modified-site /note= “Any amino acid” Modified-site /note=“A phosphorylated serine” Modified-site /note= “Any amino acid” 25 ArgXaa Arg Ser Xaa Xaa Xaa Pro 1 5 15 amino acids amino acid linear peptideunknown Modified-site /note= “A phosphorylated serine” 26 Leu Ser GlnAla Gln Ala Ser Thr Xaa Thr Pro Asn Val His Ala 1 5 10 15 15 amino acidsamino acid linear peptide unknown Modified-site /note= “A phosphorylatedserine” 27 Leu Ser Gln Ala Gln Arg Ser Thr Xaa Thr Pro Asn Val His Ala 15 10 15 15 amino acids amino acid linear peptide unknown Modified-site/note= “A phosphorylated serine” 28 Leu Ser Gln Arg Gln Ala Ser Thr XaaThr Pro Asn Val His Ala 1 5 10 15 15 amino acids amino acid linearpeptide unknown Modified-site /note= “A phosphorylated serine” 29 LeuSer Gln Ala Gln Ser Arg Thr Xaa Thr Pro Asn Val His Ala 1 5 10 15 15amino acids amino acid linear peptide unknown Modified-site /note= “Aphosphorylated serine” 30 Leu Ser Gln Ala Arg Gln Ser Thr Xaa Thr ProAsn Val His Ala 1 5 10 15 15 amino acids amino acid linear peptideunknown Modified-site /note= “A phosphorylated serine” 31 Leu Ser GlnArg Gln Arg Ser Thr Xaa Thr Ala Asn Val His Met 1 5 10 15 6 amino acidsamino acid linear peptide unknown Modified-site /note= “Any amino acid”Modified-site /note= “Any amino acid” 32 Arg Ser Xaa Ser Xaa Pro 1 5 15amino acids amino acid linear peptide unknown Modified-site /note= “Anyamino acid” Modified-site /note= “A phosphorylated serine” 33 Leu ProLys Xaa Asn Arg Ser Ala Xaa Glu Pro Ser Leu His Arg 1 5 10 15 15 aminoacids amino acid linear peptide unknown Modified-site /note= “Any aminoacid” Modified-site /note= “A phosphorylated serine” 34 Leu Pro Lys IleAsn Xaa Ser Ala Xaa Glu Pro Ser Leu His Arg 1 5 10 15 16 amino acidsamino acid linear peptide unknown Modified-site /note= “Any amino acid”Modified-site 10 /note= “A phosphorylated serine” 35 Leu Pro Lys Ile AsnArg Ser Xaa Ala Xaa Glu Pro Ser Leu His Arg 1 5 10 15 15 amino acidsamino acid linear peptide unknown Modified-site /note= “Any amino acid”Modified-site /note= “A phosphorylated serine” 36 Leu Pro Lys Ile AsnArg Ser Xaa Xaa Glu Pro Ser Leu His Arg 1 5 10 15 15 amino acids aminoacid linear peptide unknown Modified-site /note= “A phosphorylatedserine” Modified-site 10 /note= “Any amino acid” 37 Leu Pro Lys Ile AsnArg Ser Ala Xaa Xaa Pro Ser Leu His Arg 1 5 10 15 15 amino acids aminoacid linear peptide unknown Modified-site /note= “A phosphorylatedserine” Modified-site 11 /note= “Any amino acid” 38 Leu Pro Lys Ile AsnArg Ser Ala Xaa Glu Xaa Ser Leu His Arg 5 10 15 6 amino acids amino acidlinear peptide unknown 39 Arg Ser Thr Ser Thr Pro 1 5 6 amino acidsamino acid linear peptide unknown 40 Arg Ser Ala Ser Glu Pro 1 5 6 aminoacids amino acid linear peptide unknown 41 Arg Ser Ser Ser Ala Pro 1 5 6amino acids amino acid linear peptide unknown 42 Arg Ser His Ser Tyr Pro1 5 6 amino acids amino acid linear peptide unknown 43 Arg Ser Pro SerMet Pro 1 5 6 amino acids amino acid linear peptide unknown 44 Arg SerLys Ser Ala Pro 1 5 6 amino acids amino acid linear peptide unknown 45Arg Ser Pro Ser Ser Pro 1 5 6 amino acids amino acid linear peptideunknown 46 Arg Ser Gln Ser Gln Asn 1 5 6 amino acids amino acid linearpeptide unknown 47 Arg His Ala Ser Ser Pro 1 5 6 amino acids amino acidlinear peptide unknown Modified-site /note= “A phosphorylated serine” 48Arg Ser Arg Xaa Ala Pro 1 5 6 amino acids amino acid linear peptideunknown Modified-site /note= “A phosphorylated serine” 49 Arg Ser CysXaa Ile Pro 1 5 6 amino acids amino acid linear peptide unknownModified-site /note= “A phosphorylated serine” 50 Arg Ser Thr Xaa ArgPro 1 5 6 amino acids amino acid linear peptide unknown Modified-site/note= “A phosphorylated serine” 51 Arg Ser Pro Xaa Phe Pro 1 5 6 aminoacids amino acid linear peptide unknown Modified-site /note= “Aphosphorylated serine” 52 Arg Ser Glu Xaa Ser Pro 1 5 6 amino acidsamino acid linear peptide unknown Modified-site /note= “A phosphorylatedserine” 53 Arg Ser Pro Xaa Ile Pro 1 5 6 amino acids amino acid linearpeptide unknown Modified-site /note= “A phosphorylated serine” 54 ArgSer Pro Xaa Lys Pro 1 5 6 amino acids amino acid linear peptide unknownModified-site /note= “A phosphorylated serine” 55 Arg Ser Pro Xaa GlyPro 1 5 6 amino acids amino acid linear peptide unknown Modified-site/note= “A phosphorylated serine” 56 Arg Ser Val Xaa Ser Pro 1 5 6 aminoacids amino acid linear peptide unknown Modified-site /note= “Aphosphorylated serine” 57 Arg Ser Arg Xaa Tyr Pro 1 5 6 amino acidsamino acid linear peptide unknown Modified-site /note= “A phosphorylatedserine” 58 Arg Ser Gln Xaa Lys Pro 1 5

What is claimed is:
 1. A method for identifying a pharmacologic agentthat inhibits the activation of a signaling protein of the type thatbinds to a 14-3-3 protein, the method comprising: forming a mixturecomprising a construct containing a 14-3-3 protein or derivative thereofwhich binds to full-length Raf-1, wherein the protein or derivative isimmobilized to a substrate, a labeled peptide of about 6 to about 30amino acids containing a sequence derived from a Raf-1 sequencesurrounding serine-259 or serine-621, where the serine residues in thepeptide that correspond to serine-259 and serine-621 are phosphorylated,and said pharmacologic agent; subjecting the mixture to conditions underwhich presence of the pharmacologic agent decreases the binding of thelabeled peptide to the construct; and detecting the binding of thelabeled peptide to the construct wherein a decrease in binding of thelabeled peptide indicates a binding of the pharmacologic agent to theconstruct.
 2. The method of claim 1 wherein the peptide contains asequence as set forth in a member selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:
 3. 3. The method of claim 2wherein the peptide contains the sequence as set forth in SEQ ID NO: 5or SEQ ID NO:
 10. 4. The method of claim 3 wherein the signaling proteinis Raf-1.
 5. The method of claim 1 wherein the labeled peptide isradioactive.
 6. The method of claim 1 wherein the label in the labeledpeptide comprises an enzyme or a fluorescer.
 7. The method of claim 1wherein the peptide contains a sequence selected from the groupconsisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ IDNO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQID NO: 57, and SEQ ID NO:
 58. 8. The method of claim 1 wherein thepharmacologic agent is effective in inhibiting growth or differentiationof a cell.
 9. The method of claim 1 wherein the pharmacologic agent iseffective in treating cancer, atherosclerosis, or autoimmune disease.10. A method for identifying a pharmacologic agent that inhibits theactivation of a signaling protein of the type that binds to a 14-3-3protein, the method comprising: forming a mixture comprising a constructcontaining a peptide of about 6 to about 30 amino acids containing asequence derived from a Raf-1 sequence surrounding serine-259 orserine-621 immobilized to a substrate, where the serine residues in thepeptide that correspond to serine-259 and serine-621 are phosphorylated,a labeled 14-3-3 protein or derivative thereof which binds tofull-length Raf-1, and said pharmacologic agent; subjecting the mixtureto conditions under which presence of the pharmacologic agent decreasesthe binding of the construct to the labeled 14-3-3 protein or derivativethereof; and detecting the binding of the construct to the labeled14-3-3 protein or derivative thereof wherein a decrease in binding ofthe labeled 14-3-3 protein or derivative thereof indicates a binding ofthe pharmacologic agent to the 14-3-3 protein or derivative thereof. 11.The method of claim 10 wherein the peptide contains a sequence as setforth in a member selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO:
 3. 12. The method of claim 11 wherein thepeptide contains the sequence as set forth in SEQ ID NO:5 or SEQ ID NO:10.
 13. The method of claim 12 wherein the signaling protein is Raf-1.14. The method of claim 10 wherein the labeled 14-3-3 protein orderivative thereof is radioactive.
 15. The method of claim 10 whereinthe label in the labeled 14-3-3 protein or derivative thereof comprisesan enzyme or a fluorescer.
 16. The method of claim 10 wherein thepeptide contains a sequence selected from the group consisting of SEQ IDNO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 48, SEQID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53,SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ IDNO:
 58. 17. The method of claim 10 wherein the pharmacologic agent iseffective in inhibiting growth or differentiation of a cell.
 18. Themethod of claim 10 wherein the pharmacologic agent is effective intreating cancer, atherosclerosis, or autoimmune disease.
 19. A methodfor identifying a pharmacologic agent that inhibits the activation of asignaling protein, the method comprising: forming a mixture comprising a14-3-3 protein or derivative thereof which binds to full-length Raf-1, apeptide containing a sequence derived from a Raf-1 sequence surroundinga phosphorylated serine-259 or a phosphorylated serine-621, and saidpharmacologic agent, where either the 14-3-3 protein or derivativethereof or the peptide is immobilized to a substrate to form aconstruct, and where the non-immobilized entity is labeled; subjectingthe mixture to conditions under which presence of the pharmacologicagent decreases the binding of the labeled entity to the construct; anddetecting the binding of the labeled entity to the construct wherein adecrease in binding of the labeled entity indicates a binding of thepharmacologic agent to the 14-3-3 protein or derivative thereof.
 20. Themethod of claim 19 wherein the peptide contains a sequence as set forthin a member selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3 and derivatives thereof.
 21. The method of claim 20wherein the peptide contains the sequence as set forth in SEQ ID NO: 5or SEQ ID NO:
 10. 22. The method of claim 21 wherein the signalingprotein is Raf-1.
 23. The method of claim 19 wherein the labeled entityis radioactive.
 24. The method of claim 19 wherein the label in thelabeled entity comprises an enzyme or a fluorescer.
 25. The method ofclaim 19 wherein the peptide contains a sequence selected from the groupconsisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ IDNO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQID NO: 57, and SEQ ID NO:
 58. 26. The method of claim 19 wherein thepharmacologic agent is effective in inhibiting growth or differentiationof a cell.
 27. The method of claim 19 wherein the pharmacologic agent iseffective in treating cancer, atherosclerosis, or autoimmune disease.